Summary Mutations in the X-linked gene, methyl-CpG binding protein 2 (Mecp2), underlie a wide range of neuropsychiatric disorders, most commonly, Rett syndrome (RTT) - a severe autism spectrum disorder that affects about one in 10,000 female live births. Although it is well established that MeCP2 dysfunction underlies most cases of RTT, the loss of the specific MeCP2 function, which leads to RTT neuropathology remains enigmatic. MeCP2 is a member of the methyl-CpG-binding protein family that acts as transcriptional repressors. Unexpectedly, however, transcriptional profiling of brains of symptomatic RTT mice reveals only subtle changes. Furthermore, our recent studies show that inducible postnatal loss of MeCP2 in mice results in dramatic reduction in the levels of several synaptic proteins, but not their corresponding mRNAs. Together, these observations raise a possibility that the loss of a MeCP2 function different from regulation of transcription may mediate RTT. In support of this view, previous in vitro studies have shown that MeCP2 regulates splicing of minigenes; however, whether MeCP2 regulates alternative splicing in vivo remains unknown. We hypothesize that MeCP2 is an activity-dependent alternative RNA processing regulator and that its loss in RTT brain mediates aberration in alternative RNA processing during experience-driven neuronal activity, resulting in dysfunction of the mature neuronal networks. We propose to explore this potential function of MeCP2 ex-vivo and in vivo in the following two specific aims: In Aim 1, we will analyze alternative RNA synthesis and processing events in wild type and RTT neuronal cultures at rest and after neuronal activity. We will use a high throughput RNA-sequencing (RNA-seq) to identify the full sets of differential RNA synthesis and processing events regulated in unstimulated (rest) and upon activity of mature cortical neuronal cultures from wild type and RTT brains. We will focus on three main types of alternative RNA synthesis and processing events: a) alternative promoter usage, b) alternative polyadenylation, c) most importantly, alternative splicing. Both mRNAs as well as microRNAs (miRNAs) will be analyzed. In Aim 2, we will analyze alternative RNA synthesis and processing at rest and upon neuronal activity in normal and RTT mouse brains. We will examine three different brain areas; the hippocampus, prefrontal cortex, and visual cortex, and compare alternative RNA synthesis and processing in an unstimulated (at rest) and stimulated (upon activity) wild type and RTT brains, using RNA-seq. As in Aim 1, we will focus on three types of alternative RNA synthesis and processing events: a) differential promoter usage, b) alternative polyadenylation, c) alternative splicing. These aims will lay the foundation for the next step of this project - investigation of the mechanism by which MeCP2 affects alternative RNA processing and whether MeCP2 has a direct or indirect role in this process. Together, these studies will afford important insights into the function of MeCP2 in the brain and into the mechanism underlying RTT, and have the potential to highlight a target for therapeutic intervention of this devastating neurological disorder.